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Genetic toxicity in vitro

Description of key information

Ames test: negative

Mammalian cell gene mutation assay/ HPRT test: negative

Link to relevant study records

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2009 - Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 2016
GLP compliance:
yes (incl. certificate)
Remarks:
Quality Assurance statement, 4 May 2010
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: DEJ30990H0SOURCE OF TEST MATERIAL
- Analytical purity: >= 99.9 %
- Expiration date of the lot/batch: 30 Oct. 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25°C
- Solubility and stability of the test substance in the solvent/vehicle: preliminary solubility tests revealed limit concentration of 906.8 µg/mL in culture medium, solubility in DMSO of at least 90.68 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solution preperation under subdued lighting by formulating the test article in DMSOn(vortex mixing if required); The solutions were protected from light and used within 2 hours


Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, USA; Sprague Dawley rats induced with Aroclor 1254
- method of preparation of S9 mix : G6P (180 mg/mL), NADP (25 mg/mL), KCl (150mM) and S9 were mixed in ratio 1:1:1:2
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9-mix to each cell culture (19 mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test, protein content analysis, test for presence of adventitious agents, promutagen activation
Test concentrations with justification for top dose:
Range finder: 22.86; 45.71; 91.43; 182.9; 365.7; 731.4 µg/mL (with and without S-9 mix)
Experiment 1: 100, 200, 300, 350, 400, 450, 500, 550, 650, 731.4 (without S-9 mix)
100, 200, 300, 400, 500, 550, 600, 650, 700, 731.4 (with S-9 mix)
Experiment 2: 50, 100, 200, 300, 350, 400, 450, 500, 600, 731.4 (without S-9 mix)
100, 200, 300, 400, 450, 500, 550, 600, 650, 731.4 (with S-9 mix)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
4-nitroquinoline 1-oxide: 0.10/0.15 µg/ml (without S-9 mix); Benzo[a]pyrene: 2.00/3.00 µg/ml (with S-9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days

SELECTION AGENT (mutation assays): 6GT, 15 µg/ml

NUMBER OF REPLICATIONS: 2 (single cultures used for the positive control treatments)

Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above are considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 731.4 µg/mL, were tested in the absence and presence of S 9. Following the treatment incubation period, the highest two concentrations in the absence of S-9 and the highest three concentrations in the presence of S-9 (650 to 731.4 µg/mL in each case) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest two remaining concentrations in the absence of S-9 (500 and 550 µg/mL) and the highest remaining concentration in the presence of S 9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 450 µg/mL in the absence of S 9 and 550 µg/mL in the presence of S 9, which gave 10% and 7% RS, respectively. In the presence of S-9, no concentration gave 10 20% RS. Cultures treated at 500 and 550 µg/mL gave 21% and 7% RS, respectively, therefore both concentrations were analysed.

In Experiment 2, ten concentrations, ranging from 50 to 731.4 µg/mL in the absence of S-9 and from 100 to 731.4 µg/mL in the presence of S 9, were tested. Following the treatment incubation period, the highest two concentrations in the absence of S-9 (600 and 731.4 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S 9 (500 µg/mL) and the highest four concentrations in the presence of S 9 (550 to 731.4 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. However, the concentration of 300 µg/mL in the presence of S-9 was later rejected from analysis due to extreme heterogeneity for viability. Marked heterogeneity (also for viability) was observed at concentrations of 50 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, but these were included for comparative purposes. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 10% and 13% RS, respectively.

Tab. 1 Summary of mutation data experiment 1

Experiment 1 (3 hours treatment in the absence and presence of S-9 mix)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

4.80

 

0

 

100

3.57

 

100

 

72

3.25

NS

100

 

96

3.14

NS

200

 

74

4.91

NS

200

 

69

2.75

NS

300

 

67

2.03

NS

300

 

62

3.91

NS

350

 

43

5.24

NS

400

 

62

4.65

NS

400

 

29

7.07

NS

500

 

21

2.87

NS

450

 

10

6.85

NS

550

 

7

6.33

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

44

44.33

 

2

 

52

21.21

 

0.15

 

51

56.73

 

3

 

23

56.04

 

 

 

 

 

 

 

 

 

 

 

 

 

§: 6TG resistant mutants/106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

Tab. 2 Summary of mutation data experiment 2

Experiment 2 (3 hours treatment in the absence and presence of S-9 mix)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.59

 

0

 

100

2.49

 

50

$$

80

1.85

 

100

 

127

3.84

NS

100

 

71

2.38

NS

200

 

76

3.36

NS

200

 

68

3.50

NS

400

 

45

2.70

NS

300

 

48

2.28

NS

450

$$

30

2.96

 

350

 

27

4.48

NS

500

 

13

5.28

NS

400

 

18

3.32

NS

 

 

 

 

 

450

 

10

10.10

*

 

 

 

 

 

Linear trend

 

*

Linear trend

 

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

61

24.70

 

2

 

58

39.09

 

0.15

 

41

21.20

 

3

 

32

69.84

 

 

 

 

 

 

 

 

 

 

 

 

 

§: 6TG resistant mutants/ 106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

* : Comparison of each treatment with control: Dunnett#s test (one-sided), significant at 5% level

*,**,*** : Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

$$ : Treatment has marked heterogeneity for viability but is included for comparative purposes

Conclusions:
The test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under the tested conditions.
Executive summary:

The test substance was investigated for mutation at the hprt locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by two independent experiments (with and without rat S9 metabolic activation). The test article was formulated in DMSO. In the range-finding study concentrations of 22.86 - 731.4 µg/mL were tested. The highest concentration to provide more than 10% survival was 365.7 µg/mL. In experiment 1 ten concentrations (100 - 731.4µg/mL) were tested in the absence and presence of metabolic activation. Seven days after treatment a concentration of 450 µg/mL without S9-mix and 550 µg/mL with S9-mix gave 10% and 7% relative survival, respectively. In experiment 2, concentrations of 50 - 731.4 µg/mL without S9 -mix and 100 - 731.4 µg/mL with S9 mix were tested. Relative survival of 10% and 13% were determined for 450 µg/mL with S9-mix and 500 µg/mL without S9-mix, respectively. Positive and negative control data indicated the study validity. Testing with concentrations up to toxicity (450 µg/mL) in the abscence of S9 in experiment 2 revealed a significant increase in mutant frequency. However, the result was not reproducable with experiment 1and according to the acceptance criteria not of biological relevance. In conclusion, the test substance did not induce mutation at the hprt locus under the test conditions employed in this study.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Acceptable, well documented NTP study, which meets general standards.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
yes
Remarks:
Only Salmonella strains tested, 4 tested bacteria strains, 2-Aminoanthracene as sole indicator of efficacy of the S9-mix
Principles of method if other than guideline:
Testing was performed as reported by Mortelmans et al. (1986).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 99 %
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat and hamster S9-Mix (containing 10% Aroclor 1254-induced S9)
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 3333, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: without S9: 4-nitro-o-phenylenediamine (TA98 ); with S9: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days


NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates and was done in a replicate.
Evaluation criteria:
Positive, if a reproducible dose related response over the solvent control was obtained.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 3333 µg/plate onwards in all strains tested
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Tab. 1 Mutagenicity in bacterial tester strains

Strain: TA1535

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

0     

31

0.7

37

6.4

42

3.6

14

2.3

41

5.5

23

3.8

33     

 

 

26

4

 

 

12

2.1

 

 

17

1.2

100     

22

2.1

25

0.9

47

1.2

13

0.7

40

6.1

12

1.8

333     

21

3.4

25

0.3

41

7.5

7

1.3

36

2.2

13

1.9

1000     

31

3.3

19

0.7

55

2.6

10

2.2

42

6.2

17

5.5

3333     

0s

0

20

4

30s

15.3

7

0.7

31

3.5

14

4.7

10000     

0s

0

 

 

0t

0

 

 

0s

0

 

 

Positive Control

443

29.1

399

18

645

25.2

563

28

331

13.7

266

33.5

 

Strain: TA100

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

0     

108

3.2

101

8.7

134

8.3

117

10.4

116

7.8

95

1.5

33     

 

 

122

9.3

 

 

112

2.5

 

 

106

3

100     

158

14.7

109

3.7

160

9.1

119

4.7

152

4.3

111

3.3

333     

144

6.9

121

6.7

142

9

116

13.5

141

4.2

119

15.2

1000     

153

4.8

121

10.4

146

8

123

13.3

141

2.9

104

11.4

3333     

0s

0

111

8.4

104s

52.1

97

12.5

116

17.9

102

3.7

10000     

0t

0

 

 

t

 

 

 

0t

0

 

 

Positive Control

467

18

477

6.4

2355

34.5

1511

49.9

846

26.4

820

36.8

 

Strain: TA98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

0     

28

2.2

21

3.8

48

5.8

28

2.6

37

2.3

36

3.2

33     

 

 

20

2.3

 

 

23

2.4

 

 

31

4.9

100     

19

0.7

15

1.5

46

7

30

4.2

43

1.2

30

1.7

333     

16

1.5

17

2.3

52

0.7

24

2.6

34

4.7

31

5.2

1000     

15

0.6

20

2.1

57

2.6

22

2.7

37

6.1

35

4.1

3333     

1s

0.7

17

0.9

0s

0

23

1.5

43

4.2

26

0.3

10000     

0s

0.3

 

 

t

 

 

 

0s

0

 

 

Positive Control

758

14.2

722

8.2

1856

19.6

1102

66.6

436

5.1

591

44.4

 

Strain: TA1537

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

10% HLI
(Negative)

10% RLI
(Negative)

10% RLI
(Negative)

ug/Plate

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

Mean ± SEM

0     

11

1.8

7

1.5

19

4.7

9

1.3

14

1.2

11

1

33     

 

 

7

1.2

 

 

6

1.5

 

 

9

0

100     

5

0.9

7

0.3

23

2.8

7

0.9

10

2.7

17

1.2

333     

7

2

9

1.5

27

1

5

0.6

13

2.9

13

3.5

1000     

6

0

10

1

21

6.7

10

2.1

13

0.7

15

1.8

3333     

0s

0

7

1.2

0s

0

5s

0.9

6

1.5

12

0.3

10000     

0s

0

 

 

t

 

 

 

0s

0

 

 

Positive Control

388

33.5

205

40.4

591

16.8

465

15.2

266

9.8

241

15.6

RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination
Conclusions:
Under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive controls showed the expected values.
Executive summary:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 33, 100, 333, 1000, 3333, and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: NTP standard protocol - contains 5th strain for Ames test Key-study ( E. coli WP2 uvr A pKM 101)
Principles of method if other than guideline:
Testing was performed as reported by Mortelmans et al. (1986)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 99 %
Target gene:
Histidine
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
rat S9-Mix (10% Aroclor 1254-induced)
Test concentrations with justification for top dose:
S. typhimurium TA 98, TA 100: 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate
E. coli pKM 101: 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: TA98: 4-nitro-o-phenylenediamine, E.coli WP2 uvrA: methyl methanesulfonate, all strains: 2-aminoanthracene with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days


NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates and was done in a replicate.
Evaluation criteria:
Positive if a reproducible dose related response over the solvent control was obtained .
Species / strain:
other: S. typhimurium TA 98, TA 100, E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 1000 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tab. 1 Mutagenicity in bacterial tester strains

Strain: eColi pKM101

Dose

NA
(Negative)

10% RLI
(Negative)

NA
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

132

9.1

157

2.4

122

6.7

139

3.3

50     

186

13.9

 

 

132

9.1

155

5.5

100     

152

14.2

 

 

158

12.5

172

10.5

500     

136

4.6

201

11.9

140

3.3

151

5

1000     

105

4.8

199

30.6

67

0.3

161

7.2

1500     

150

12.2

 

 

 

 

 

 

2000     

18

8.6

182

4.6

0

0

178

14.4

3000     

 

 

110

8.5

 

 

 

 

4000     

 

 

0

0

 

 

 

 

Positive Control

1101

54.8

734

6.9

812

14.4

711

30.7

 

Strain: TA100

Dose

10% RLI
(Negative)

NA
(Negative)

10% RLI
(Negative)

NA
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

84

2.6

61

4

56

4.4

51

6

50     

90

6.7

63

6.6

51

6.4

65

2.1

100     

105

10.3

55

2.1

66

0.9

63

7

250     

 

 

 

 

 

 

64

1.9

500     

90

4.3

54

3.3

57

4

48

4

750     

 

 

 

 

 

 

55

4.7

1000     

76

0.9

t

 

47

5.8

 

 

2000     

83

7.5

t

 

63

8.5

 

 

Positive Control

768

36.1

553

13.8

609

29.4

609

28.8

 

Strain: TA98

Dose

NA
(Negative)

10% RLI
(Negative)

NA
(Negative)

10% RLI
(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

20

0.7

30

0.9

20

2.4

25

2.7

50     

17

1.2

40

2.4

17

1.2

27

1.9

100     

19

0.3

29

2

15

1

22

1.5

250     

 

 

 

 

12

3.7

 

 

500     

24

1.2

38

2.1

13

2.3

29

3.8

750     

 

 

 

 

13

0.9

 

 

1000     

t

 

36

4

 

 

19

2.1

2000     

t

 

27

0.9

 

 

25

3.5

Positive Control

417

14.8

752

58.3

572

8.4

1137

18.8

RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination
Conclusions:
Under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive controls showed the expected values.
Executive summary:

In a second Ames test conducted under NTP standard conditions (NTP, 2005), S. typhimurium TA 98 and TA 100 strains were treated with doses of 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate and E. coli WP2 uvr A pKM 101 with 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate. The results confirm the negative mutagenic potential of the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Test: negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Dec 2002 - Mar 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Peripheral blood samples were obtained from male and female B6C3F1 mice at the end of a 14-week toxicity study. Smears were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Slides were scanned at 630 or 1000 times magnification using a semi-automated image analysis system to determine the frequency of micronuclei in 10000 normochromatic erythrocytes (NCEs) in each of 10 animals per dose group. A detailed discussion of this assay can be found in MacGregor et al. (1990).
GLP compliance:
no
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Physical state: colorless liquid with a strong ammonia odor
- Analytical purity: approximately 99.9%
- Lot/batch No.: BE/07/01
- Stability under test conditions: no degradation of the bulk chemical was detected
- Storage condition of test material: at controlled room temperature
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 23.3 g (mean), female: 19.8 g (mean)
- Housing: individually in stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly and rotated daily
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure periods
- Water: Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C (72°±3 °F)
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h
Duration of treatment / exposure:
14 weeks (93 days exposure)
Frequency of treatment:
6 hours per day, 5 days per week
Dose / conc.:
8 ppm (nominal)
Remarks:
analytical concentration 8 ± 0.3 ppm
Dose / conc.:
16 ppm (nominal)
Remarks:
analytical concentration 15.9 ± 0.6 ppm
Dose / conc.:
32 ppm (nominal)
Remarks:
analytical concentration 32 ± 1.3 ppm
Dose / conc.:
62 ppm (nominal)
Remarks:
analytical concentration 62.2 ± 2.3 ppm
Dose / conc.:
125 ppm (nominal)
Remarks:
analytical concentration 126 ± 5 ppm
No. of animals per sex per dose:
5
Control animals:
yes
Tissues and cell types examined:
Peripheral blood
Statistics:
The frequency of micronucleated cells among normochromatic cells was analysed by statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test followed by pairwise comparisons between each exposure group and the control group.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified

Tab. 1 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes

Dose (ppm)

Micronucleated Normochromatic Erythrocytes / 1000 cells

 

Male

Female

0

2.80 ± 0.30

2.60 ± 0.29

8

4.60 ± 0.60

2.50 ± 0.61

16

4.10 ± 0.48

2.20 ± 0.25

32

3.30 ± 0.34

3.50 ± 0.57

62

4.00 ± 0.52

3.80 ± 0.60

125

2.60 ± 0.33

2.20 ± 0.25

No significant increase in micronucleated NCEs was observed in males or females and all tested dose groups.

Conclusions:
The percentage of polychromatic erythrocytes was unaltered, suggesting a lack of bone marrow toxicity.
Executive summary:

In a micronucleus assay with B6C3F1 mice, peripheral blood samples were obtained from 5 male/female animals at the end of a 93 days inhalation toxicity study (6 hours per day, 5 days per week; doses of 0, 8, 16, 32, 62, 125 ppm). No significant increase in micronucleated normochromatic erythrocytes was observed in males or females and all tested dose groups.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay (NTP 1987) using a standard protocol approved by the National Toxicology Program. Doses of 0, 33, 100, 333, 1000, 3333, and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate. In a second Ames test (NTP 2005) performed under NTP standard conditions, S. typhimurium TA 98 and TA 100 strains were treated with doses of 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate and E. coli WP2 uvr A pKM 101 with 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate confirmed these negative results. Further standard Ames tests with two strains or three strains revealed no evidence of mutagenicity in the bacterium Salmonella typhimurium either in the presence or absence of a mammalian liver metabolic activation system.

One negative GLP-compliant in vitro Mammalian Cell Gene Mutation Test according to OECD TG 476 is also available. The study was conducted in mammalian cells (L5178Y cells) at a test substance concentration range from 45.71 to 731.4 µg/ml with or without S-9 mix (2010, reliability 1).

 

In vivo study:

In a micronucleus assay with B6C3F1 mice, peripheral blood samples were obtained from 5 male/female animals at the end of a 13-week inhalation toxicity study (6 hours per day, 5 days per week; doses of 0, 8, 16, 32, 62, 125 ppm). No significant increase in micronucleated erythrocytes was observed in males or females and all tested dose groups.


Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity was observed in in-vitro or in-vivo studies.

As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480